Pathogenesis of SARS-CoV-2 in Transgenic Mice Expressing Human Angiotensin-Converting Enzyme 2.

COVID-19 has spread worldwide since 2019 and is now a severe threat to public health. We previously identified the causative agent as a novel SARS-related coronavirus (SARS-CoV-2) that uses human angiotensin-converting enzyme 2 (hACE2) as the entry receptor. Here, we successfully developed a SARS-CoV-2 hACE2 transgenic mouse (HFH4-hACE2 in C3B6 mice) infection model. The infected mice generated typical interstitial pneumonia and pathology that were similar to those of COVID-19 patients. Viral quantification revealed the lungs as the major site of infection, although viral RNA could also be found in the eye, heart, and brain in some mice. Virus identical to SARS-CoV-2 in full-genome sequences was isolated from the infected lung and brain tissues. Last, we showed that pre-exposure to SARS-CoV-2 could protect mice from severe pneumonia. Our results show that the hACE2 mouse would be a valuable tool for testing potential vaccines and therapeutics.

Ribosome-rescuer PELO catalyzes the oligomeric assembly of NOD-like receptor family proteins via activating their ATPase enzymatic activity.

NOD-like receptors (NLRs) are pattern recognition receptors for diverse innate immune responses. Self-oligomerization after engagement with a ligand is a generally accepted model for the activation of each NLR. We report here that a catalyzer was required for NLR self-oligomerization. PELO, a well-known surveillance factor in translational quality control and/or ribosome rescue, interacted with all cytosolic NLRs and activated their ATPase activity. In the case of flagellin-initiated NLRC4 inflammasome activation, flagellin-bound NAIP5 recruited the first NLRC4 and then PELO was required for correctly assembling the rest of NLRC4s into the NLRC4 complex, one by one, by activating the NLRC4 ATPase activity. Stoichiometric and functional data revealed that PELO was not a structural constituent of the NLRC4 inflammasome but a powerful catalyzer for its assembly. The catalytic role of PELO in the activation of cytosolic NLRs provides insight into NLR activation and provides a direction for future studies of NLR family members.

Phase-dependent growth of Pt on MoS2 for highly efficient H2 evolution.

Crystal phase is a key factor determining the properties, and hence functions, of two-dimensional transition-metal dichalcogenides (TMDs)1,2. The TMD materials, explored for diverse applications3-8, commonly serve as templates for constructing nanomaterials3,9 and supported metal catalysts4,6-8. However, how the TMD crystal phase affects the growth of the secondary material is poorly understood, although relevant, particularly for catalyst development. In the case of Pt nanoparticles on two-dimensional MoS2 nanosheets used as electrocatalysts for the hydrogen evolution reaction7, only about two thirds of Pt nanoparticles were epitaxially grown on the MoS2 template composed of the metallic/semimetallic 1T/1T' phase but with thermodynamically stable and poorly conducting 2H phase mixed in. Here we report the production of MoS2 nanosheets with high phase purity and show that the 2H-phase templates facilitate the epitaxial growth of Pt nanoparticles, whereas the 1T' phase supports single-atomically dispersed Pt (s-Pt) atoms with Pt loading up to 10 wt%. We find that the Pt atoms in this s-Pt/1T'-MoS2 system occupy three distinct sites, with density functional theory calculations indicating for Pt atoms located atop of Mo atoms a hydrogen adsorption free energy of close to zero. This probably contributes to efficient electrocatalytic H2 evolution in acidic media, where we measure for s-Pt/1T'-MoS2 a mass activity of 85 ± 23 A [Formula: see text] at the overpotential of -50 mV and a mass-normalized exchange current density of 127 A [Formula: see text] and we see stable performance in an H-type cell and prototype proton exchange membrane electrolyser operated at room temperature. Although phase stability limitations prevent operation at high temperatures, we anticipate that 1T'-TMDs will also be effective supports for other catalysts targeting other important reactions.

A Non-canonical PDK1-RSK Signal Diminishes Pro-caspase-8-Mediated Necroptosis Blockade.

Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis.

Roton pair density wave in a strong-coupling kagome superconductor.

The transition metal kagome lattice materials host frustrated, correlated and topological quantum states of matter1-9. Recently, a new family of vanadium-based kagome metals, AV3Sb5 (A = K, Rb or Cs), with topological band structures has been discovered10,11. These layered compounds are nonmagnetic and undergo charge density wave transitions before developing superconductivity at low temperatures11-19. Here we report the observation of unconventional superconductivity and a pair density wave (PDW) in CsV3Sb5 using scanning tunnelling microscope/spectroscopy and Josephson scanning tunnelling spectroscopy. We find that CsV3Sb5 exhibits a V-shaped pairing gap Δ ~ 0.5 meV and is a strong-coupling superconductor (2Δ/kBTc ~ 5) that coexists with 4a0 unidirectional and 2a0 × 2a0 charge order. Remarkably, we discover a 3Q PDW accompanied by bidirectional 4a0/3 spatial modulations of the superconducting gap, coherence peak and gap depth in the tunnelling conductance. We term this novel quantum state a roton PDW associated with an underlying vortex-antivortex lattice that can account for the observed conductance modulations. Probing the electronic states in the vortex halo in an applied magnetic field, in strong field that suppresses superconductivity and in zero field above Tc, reveals that the PDW is a primary state responsible for an emergent pseudogap and intertwined electronic order. Our findings show striking analogies and distinctions to the phenomenology of high-Tc cuprate superconductors, and provide groundwork for understanding the microscopic origin of correlated electronic states and superconductivity in vanadium-based kagome metals.

IL-27 signalling promotes adipocyte thermogenesis and energy expenditure.

Thermogenesis in brown and beige adipose tissue has important roles in maintaining body temperature and countering the development of metabolic disorders such as obesity and type 2 diabetes1,2. Although much is known about commitment and activation of brown and beige adipose tissue, its multiple and abundant immunological factors have not been well characterized3-6. Here we define a critical role of IL-27-IL-27Rα signalling in improving thermogenesis, protecting against diet-induced obesity and ameliorating insulin resistance. Mechanistic studies demonstrate that IL-27 directly targets adipocytes, activating p38 MAPK-PGC-1α signalling and stimulating the production of UCP1. Notably, therapeutic administration of IL-27 ameliorated metabolic morbidities in well-established mouse models of obesity. Consistently, individuals with obesity show significantly decreased levels of serum IL-27, which can be restored after bariatric surgery. Collectively, these findings show that IL-27 has an important role in orchestrating metabolic programs, and is a highly promising target for anti-obesity immunotherapy.

The KDM5A/RBP2 histone demethylase represses NOTCH signaling to sustain neuroendocrine differentiation and promote small cell lung cancer tumorigenesis.

More than 90% of small cell lung cancers (SCLCs) harbor loss-of-function mutations in the tumor suppressor gene RB1 The canonical function of the RB1 gene product, pRB, is to repress the E2F transcription factor family, but pRB also functions to regulate cellular differentiation in part through its binding to the histone demethylase KDM5A (also known as RBP2 or JARID1A). We show that KDM5A promotes SCLC proliferation and SCLC's neuroendocrine differentiation phenotype in part by sustaining expression of the neuroendocrine transcription factor ASCL1. Mechanistically, we found that KDM5A sustains ASCL1 levels and neuroendocrine differentiation by repressing NOTCH2 and NOTCH target genes. To test the role of KDM5A in SCLC tumorigenesis in vivo, we developed a CRISPR/Cas9-based mouse model of SCLC by delivering an adenovirus (or an adeno-associated virus [AAV]) that expresses Cre recombinase and sgRNAs targeting Rb1, Tp53, and Rbl2 into the lungs of Lox-Stop-Lox Cas9 mice. Coinclusion of a KDM5A sgRNA decreased SCLC tumorigenesis and metastasis, and the SCLCs that formed despite the absence of KDM5A had higher NOTCH activity compared to KDM5A+/+ SCLCs. This work establishes a role for KDM5A in SCLC tumorigenesis and suggests that KDM5 inhibitors should be explored as treatments for SCLC.

Structure of GPR101-Gs enables identification of ligands with rejuvenating potential.

GPR101 is an orphan G protein-coupled receptor actively participating in energy homeostasis. Here we report the cryo-electron microscopy structure of GPR101 constitutively coupled to Gs heterotrimer, which reveals unique features of GPR101, including the interaction of extracellular loop 2 within the 7TM bundle, a hydrophobic chain packing-mediated activation mechanism and the structural basis of disease-related mutants. Importantly, a side pocket is identified in GPR101 that facilitates in silico screening to identify four small-molecule agonists, including AA-14. The structure of AA-14-GPR101-Gs provides direct evidence of the AA-14 binding at the side pocket. Functionally, AA-14 partially restores the functions of GH/IGF-1 axis and exhibits several rejuvenating effects in wild-type mice, which are abrogated in Gpr101-deficient mice. In summary, we provide a structural basis for the constitutive activity of GPR101. The structure-facilitated identification of GPR101 agonists and functional analysis suggest that targeting this orphan receptor has rejuvenating potential.

Phase Engineering of Nanomaterials: Transition Metal Dichalcogenides.

Crystal phase, a critical structural characteristic beyond the morphology, size, dimension, facet, etc., determines the physicochemical properties of nanomaterials. As a group of layered nanomaterials with polymorphs, transition metal dichalcogenides (TMDs) have attracted intensive research attention due to their phase-dependent properties. Therefore, great efforts have been devoted to the phase engineering of TMDs to synthesize TMDs with controlled phases, especially unconventional/metastable phases, for various applications in electronics, optoelectronics, catalysis, biomedicine, energy storage and conversion, and ferroelectrics. Considering the significant progress in the synthesis and applications of TMDs, we believe that a comprehensive review on the phase engineering of TMDs is critical to promote their fundamental studies and practical applications. This Review aims to provide a comprehensive introduction and discussion on the crystal structures, synthetic strategies, and phase-dependent properties and applications of TMDs. Finally, our perspectives on the challenges and opportunities in phase engineering of TMDs will also be discussed.

Gut stem cell necroptosis by genome instability triggers bowel inflammation.

The aetiology of inflammatory bowel disease (IBD) is a multifactorial interplay between heredity and environment1,2. Here we report that deficiency in SETDB1, a histone methyltransferase that mediates the trimethylation of histone H3 at lysine 9, participates in the pathogenesis of IBD. We found that levels of SETDB1 are decreased in patients with IBD, and that mice with reduced SETDB1 in intestinal stem cells developed spontaneous terminal ileitis and colitis. SETDB1 safeguards genome stability3, and the loss of SETDB1 in intestinal stem cells released repression of endogenous retroviruses (retrovirus-like elements with long repeats that, in humans, comprise approximately 8% of the genome). Excessive viral mimicry generated by motivated endogenous retroviruses triggered Z-DNA-binding protein 1 (ZBP1)-dependent necroptosis, which irreversibly disrupted homeostasis of the epithelial barrier and promoted bowel inflammation. Genome instability, reactive endogenous retroviruses, upregulation of ZBP1 and necroptosis were all seen in patients with IBD. Pharmaceutical inhibition of RIP3 showed a curative effect in SETDB1-deficient mice, which suggests that targeting necroptosis of intestinal stem cells may represent an approach for the treatment of severe IBD.

Genomic analysis reveals a cryptic pangolin species.

Eight extant species of pangolins are currently recognized. Recent studies found that two mitochondrial haplotypes identified in confiscations in Hong Kong could not be assigned to any known pangolin species, implying the existence of a species. Here, we report that two additional mitochondrial haplotypes identified in independent confiscations from Yunnan align with the putative species haplotypes supporting the existence of this mysterious species/population. To verify the new species scenario we performed a comprehensive analysis of scale characteristics and 138 whole genomes representing all recognized pangolin species and the cryptic new species, 98 of which were generated here. Our morphometric results clearly attributed this cryptic species to Asian pangolins (Manis sp.) and the genomic data provide robust and compelling evidence that it is a pangolin species distinct from those recognized previously, which separated from the Philippine pangolin and Malayan pangolin over 5 Mya. Our study provides a solid genomic basis for its formal recognition as the ninth pangolin species or the fifth Asian one, supporting a new taxonomic classification of pangolins. The effects of glacial climate changes and recent anthropogenic activities driven by illegal trade are inferred to have caused its population decline with the genomic signatures showing low genetic diversity, a high level of inbreeding, and high genetic load. Our finding greatly expands current knowledge of pangolin diversity and evolution and has vital implications for conservation efforts to prevent the extinction of this enigmatic and endangered species from the wild.

Orexin Facilitates the Peripheral Chemoreflex via Corticotropin-Releasing Hormone Neurons Projecting to the Nucleus of the Solitary Tract.

We previously showed that orexin neurons are activated by hypoxia and facilitate the peripheral chemoreflex (PCR)-mediated hypoxic ventilatory response (HVR), mostly by promoting the respiratory frequency response. Orexin neurons project to the nucleus of the solitary tract (nTS) and the paraventricular nucleus of the hypothalamus (PVN). The PVN contributes significantly to the PCR and contains nTS-projecting corticotropin-releasing hormone (CRH) neurons. We hypothesized that in male rats, orexin neurons contribute to the PCR by activating nTS-projecting CRH neurons. We used neuronal tract tracing and immunohistochemistry (IHC) to quantify the degree that hypoxia activates PVN-projecting orexin neurons. We coupled this with orexin receptor (OxR) blockade with suvorexant (Suvo, 20 mg/kg, i.p.) to assess the degree that orexin facilitates the hypoxia-induced activation of CRH neurons in the PVN, including those projecting to the nTS. In separate groups of rats, we measured the PCR following systemic orexin 1 receptor (Ox1R) blockade (SB-334867; 1 mg/kg) and specific Ox1R knockdown in PVN. OxR blockade with Suvo reduced the number of nTS and PVN neurons activated by hypoxia, including those CRH neurons projecting to nTS. Hypoxia increased the number of activated PVN-projecting orexin neurons but had no effect on the number of activated nTS-projecting orexin neurons. Global Ox1R blockade and partial Ox1R knockdown in the PVN significantly reduced the PCR. Ox1R knockdown also reduced the number of activated PVN neurons and the number of activated tyrosine hydroxylase neurons in the nTS. Our findings suggest orexin facilitates the PCR via nTS-projecting CRH neurons expressing Ox1R.

Intracytoplasmic sperm injection versus conventional in-vitro fertilisation for couples with infertility with non-severe male factor: a multicentre, open-label, randomised controlled trial.

BACKGROUND: Introduced in 1992, intracytoplasmic sperm injection (ICSI) was initially indicated for severe male infertility; however, its use has since been expanded to non-severe male infertility. We aimed to compare the efficacy and safety of ICSI versus conventional in-vitro fertilisation (IVF) in couples with infertility with non-severe male factor. METHODS: We conducted an investigator-initiated, multicentre, open-label, randomised controlled trial in ten reproductive medicine centres across China. Couples with infertility with non-severe male factor without a history of poor fertilisation were randomly assigned (1:1) to undergo either ICSI or conventional IVF. The primary outcome was live birth after first embryo transfer. We performed the primary analysis in the intention-to-treat population using log-binomial regression models for categorical outcomes or linear regression models for continuous outcomes, adjusting for centre. This trial is registered with Clinicaltrials.gov, NCT03298633, and is completed. FINDINGS: Between April 4, 2018, and Nov 15, 2021, 3879 couples were screened, of whom 2387 (61·5%) couples were randomly assigned (1184 [49·6%] to the ICSI group and 1203 [50·4%] to the conventional IVF group). After excluding couples who were ineligible, randomised twice, or withdrew consent, 1154 (97·5%) in the ICSI group and 1175 (97·7%) in the conventional IVF group were included in the primary analysis. Live birth after first embryo transfer occurred in 390 (33·8%) couples in the ICSI group and in 430 (36·6%) couples in the conventional IVF group (adjusted risk ratio [RR] 0·92 [95% CI 0·83-1·03]; p=0·16). Two (0·2%) neonatal deaths were reported in the ICSI group and one (0·1%) in the conventional IVF group. INTERPRETATION: In couples with infertility with non-severe male factor, ICSI did not improve live birth rate compared with conventional IVF. Given that ICSI is an invasive procedure associated with additional costs and potential increased risks to offspring health, routine use is not recommended in this population. FUNDING: National Natural Science Foundation of China, National Key Research and Development Program, Beijing Municipal Science & Technology Commission, and Peking University Third Hospital.

Profiling chromatin accessibility in formalin-fixed paraffin-embedded samples.

Archived formalin-fixed paraffin-embedded (FFPE) samples are the global standard format for preservation of the majority of biopsies in both basic research and translational cancer studies, and profiling chromatin accessibility in the archived FFPE tissues is fundamental to understanding gene regulation. Accurate mapping of chromatin accessibility from FFPE specimens is challenging because of the high degree of DNA damage. Here, we first showed that standard ATAC-seq can be applied to purified FFPE nuclei but yields lower library complexity and a smaller proportion of long DNA fragments. We then present FFPE-ATAC, the first highly sensitive method for decoding chromatin accessibility in FFPE tissues that combines Tn5-mediated transposition and T7 in vitro transcription. The FFPE-ATAC generates high-quality chromatin accessibility profiles with 500 nuclei from a single FFPE tissue section, enables the dissection of chromatin profiles from the regions of interest with the aid of hematoxylin and eosin (H&E) staining, and reveals disease-associated chromatin regulation from the human colorectal cancer FFPE tissue archived for >10 yr. In summary, the approach allows decoding of the chromatin states that regulate gene expression in archival FFPE tissues, thereby permitting investigators to better understand epigenetic regulation in cancer and precision medicine.

The RNA ligase RNA terminal phosphate cyclase B regulates mRNA alternative splicing and is required for mouse oocyte development and maintenance.

Recent large-scale mRNA sequencing has shown that introns are retained in 5-10% of mRNA, and these events are named intron retention (IR). IR has been recognized as a key mechanism in the regulation of gene expression. However, the role of this mechanism in female reproduction in mammals remains unclear. RNA terminal phosphate cyclase B (RTCB) is a RNA ligase; we found that RTCB conditional knockout mice have premature ovarian failure and that RTCB plays a crucial role in follicular development. RTCB regulated the splicing of transcripts related to DNA methylation and DNA damage repair. In addition, it regulated the resumption of oocyte meiosis by affecting CDK1 activation. Moreover, the loss of RTCB suppressed zygotic genome activation (ZGA) and decreased translation at the global level. In addition, Rtcb deletion resulted in the accumulation of maternal mRNAs containing unspliced introns and in a decline in the overall level of transcripts. As a result, the Rtcb-/- females were sterile. Our study highlights the important role of RTCB-regulated noncanonical alternative splicing in female reproduction.

1T'-transition metal dichalcogenide monolayers stabilized on 4H-Au nanowires for ultrasensitive SERS detection.

Unconventional 1T'-phase transition metal dichalcogenides (TMDs) have aroused tremendous research interest due to their unique phase-dependent physicochemical properties and applications. However, due to the metastable nature of 1T'-TMDs, the controlled synthesis of 1T'-TMD monolayers (MLs) with high phase purity and stability still remains a challenge. Here we report that 4H-Au nanowires (NWs), when used as templates, can induce the quasi-epitaxial growth of high-phase-purity and stable 1T'-TMD MLs, including WS2, WSe2, MoS2 and MoSe2, via a facile and rapid wet-chemical method. The as-synthesized 4H-Au@1T'-TMD core-shell NWs can be used for ultrasensitive surface-enhanced Raman scattering (SERS) detection. For instance, the 4H-Au@1T'-WS2 NWs have achieved attomole-level SERS detections of Rhodamine 6G and a variety of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins. This work provides insights into the preparation of high-phase-purity and stable 1T'-TMD MLs on metal substrates or templates, showing great potential in various promising applications.

EphA2 is a functional entry receptor for HCMV infection of glioblastoma cells.

Human cytomegalovirus (HCMV) infection is associated with human glioblastoma, the most common and aggressive primary brain tumor, but the underlying infection mechanism has not been fully demonstrated. Here, we show that EphA2 was upregulated in glioblastoma and correlated with the poor prognosis of the patients. EphA2 silencing inhibits, whereas overexpression promotes HCMV infection, establishing EphA2 as a crucial cell factor for HCMV infection of glioblastoma cells. Mechanistically, EphA2 binds to HCMV gH/gL complex to mediate membrane fusion. Importantly, the HCMV infection was inhibited by the treatment of inhibitor or antibody targeting EphA2 in glioblastoma cells. Furthermore, HCMV infection was also impaired in optimal glioblastoma organoids by EphA2 inhibitor. Taken together, we propose EphA2 as a crucial cell factor for HCMV infection in glioblastoma cells and a potential target for intervention.

Persulfidation and phosphorylation of transcription factor SlWRKY6 differentially regulate tomato fruit ripening.

Cysteine desulfhydrase (LCD) catalyzes the generation of the signaling molecule hydrogen sulfide (H2S) in plants. In this study, we found that H2S can inhibit tomato (Solanum lycopersicum) fruit ripening and SlWRKY6 undergoes differential protein persulfidation in SlLCD1-overexpressing leaves. Then, further study indicated that SlWRKY6 could be persulfidated by H2S at Cys396. By construction of slwrky6 mutants and SlWRKY6-OE lines, we found that SlWRKY6 positively regulates leaf senescence and fruit ripening by activating the transcription of ripening-related genes STAYGREEN 1 (SlSGR1) and Senescence-Associated Gene 12 (SlSAG12). In addition, SlWRKY6 interacted with kinase SlMAPK4 and was phosphorylated at Ser33. Dual luciferase transient expression assays and electrophoretic mobility shift assays indicated that SlWRKY6 persulfidation attenuated its transcriptional regulation of target genes SlSGR1 and SlSAG12, whereas SlWRKY6 phosphorylation by SlMAPK4 activated the transcription of target genes to promote fruit ripening. Moreover, we provided evidence that SlWRKY6 persulfidation attenuated its SlMAPK4-mediated phosphorylation to inhibit tomato fruit ripening. By transient expression of SlWRKY6, SlWRKY6C396A, SlWRKY6S33A and SlWRKY6S33D in slwrky6 fruits, we found that SlWRKY6 persulfidation attenuated the expression of SlSGR1 and SlSAG12 thereby delaying tomato fruit ripening, while SlWRKY6 phosphorylation increased the expression of target genes. As tomato fruits ripened, endogenous H2S production decreased, while SlMAPK4 expression increased. Therefore, our findings reveal a model in which SlWRKY6 persulfidation due to higher endogenous H2S levels in un-ripened fruit inhibits its ability to activate SlSGR1 and SlSAG12 expression, while SlWRKY6 phosphorylation by SlMAPK4 activates its transcriptional activity, thereby promoting tomato fruit ripening.

The effectiveness of expanded carrier screening based on next-generation sequencing for severe monogenic genetic diseases.

Expanded carrier screening (ECS) based on next-generation sequencing has been the subject of few studies to estimate the effectiveness of ECS in the Chinese population. A total of 3737 individuals from Southwest China or the general Chinese population, including 1048 pairs and 1641 individuals, were analysed by ECS for 155 monogenetic diseases. An ECS panel was used to detect 147 genes and 10,449 variants in 145 autosomal recessive and 10 X-linked recessive disorders. A total of 43.27% (1617/3737) were found to be carriers of at least one of the 155 monogenetic diseases. The average number of carriers of these recessive mutations was 0.54 and ranged from 0 to 4. Of the 1048 couples, 74.81% (n = 784) were found to have at least one partner carrying more than one disease. In addition, 5.34% of the couples at risk (n = 56) were heterozygous for the same autosomal recessive disease, and 0.37% of the women (9/2440) were carriers of X-linked diseases. Our study demonstrated the clinical significance of ECS in Chinese populations and the need for a programme of familial screening for the prevention of severe recessive monogenetic diseases.